Clinical assessment of mocravimod as a victim of drug–drug interactions via CYP3A4 metabolism and transporters

Dymphy R. Huntjens, Stephan Oehen, and Elisabeth Kueenburg

Abstract

Mocravimod, a novel immunomodulator targeting sphingosine-1-phosphate receptor (S1PR), is being developed as a maintenance treatment for patients with acute myeloid leukemia undergoing allogeneic hematopoietic cell transplantation. Preclinical data suggested that cytochrome (CYP) 3A4 is the primary enzyme involved in mocravimod metabolism. In vitro data showed that both mocravimod and its active metabolite mocravimod-phosphate are substrates for breast cancer resistance protein (BCRP) and P-glycoprotein (P-gp) but not for organic-anion-transporting polypeptides (OATP1B1 and OATP1B3). As mocravimod is co-administered with CYP3A4 inhibitors such as azoles or cyclosporin, the potential for drug–drug interactions (DDIs) was evaluated. In addition, the effect of BCRP, P-gp, and OATP via cyclosporin coadministration was assessed. Two open-label, two-period, fixed-sequence studies were conducted in healthy subjects to evaluate the DDI potential of mocravimod and 1) itraconazole, a dual CYP3A4/P-gP inhibitor, or 2) cyclosporin, a moderate inhibitor of CYP3A4 and P-gp, BCRP, OATP1B1, and OATP1B3. Safety and tolerability were also monitored. The PK of mocravimod and mocravimod-phosphate were bioequivalent with or without co-administration of multiple doses of itraconazole and a moderate interaction is observed when co-administered with cyclosporin. The most commonlyreported treatment-emergent adverse events were bradycardia and decreased lymphocyte count, which are expected side effects for S1PR modulators.

Keywords: cyclosporine, drug–drug interaction, immunomodulator, itraconazole, pharmacokinetics

S1PR modulation does not interfere with T cell cytotoxicity, hence function is maintained, and anti-leukemia response remains potent.² Mocravimod’s potential to preserve the desirable GvL effect while decreasing GvHD is a promising way to maintain the prospect of a cure while decreasing transplantrelated mortality (TRM) and morbidity in AML patients undergoing allogeneic HCT. In the current ongoing phase 3 study for the allo-HCT, several conditioning regimens are allowed such as combinations of busulphan, treosulphan, melphalan, fludarabine, clofarabine, cytarabinem etoposide, cyclophosphamide, carmustine, and/or thiotepa – that are required to eliminate the bone marrow by conditioning via high-dose chemotherapy and then proceed to stem cell transplant via donor cells.

All patients undergoing allo-HCT receive prophylaxis against fungal and bacterial infections. Anti-fungal and some antibiotic medications are CYP3A4 inhibitors. Calcineurin inhibitors, such as cyclosporine A, are key forGvHD prophylaxis in patients undergoing allo-HCT. Mocravimod (1 and 3 mg once daily, o.d.) is under study to be administered concomitantly with these standard of care treatments such as azole anti-fungals (CYP3A4 inhibitors) and immunosuppressants, for example cyclosporin in allo-HCT patients. As such, it is important to know, if there are drug–drug interactions that may impact mocravimod and/or mocravimod-phosphate exposure.

Single (0.1–40 mg) and multiple ascending-dose (0.3–3 mg o.d.) studies in healthy subjects demonstrated a favorable safety, tolerability, pharmacokinetic (PK), and pharmacodynamic (PD) profile of mocravimod and mocravimod-phosphate; linear PK was seen following single doses of mocravimod ranging from 2 to 40 mg and absorption was slow with maximum blood concentration (C_max) reached at a median time of 6 to 12 h post-dose for both mocravimod and mocravimod-phosphate. Blood concentration-time profiles of both compounds showed a slight rebound between 24 to 48 h after dosing indicating the possibility of entero-hepatic recycling.

In vitro data did not suggest that OATP1B1, 1B3, and OCT1 mediated the uptake of mocravimod and mocravimod-phosphate. However, based upon in vitro studies in Madin–Darby canine kidney cells, it was shown that both mocravimod and mocravimodphosphate are substrates for breast cancer resistance protein (BCRP) and P-glycoprotein (P-gp).

Therefore, it is necessary to investigate these potential interactions in accordance with the recommendations of the Food and Drug Administration (FDA)⁴ and the European Medicines Agency (EMA)⁵ to assess the potential DDIs⁶ of mocravimod.

Various S1P modulators, such as fingolimod, cenerimod, ponesimod, siponimod have been shown to slow the heart rate, as a result of S1P binding to multiple receptors including S1P1R.¹⁶ Furthermore, fingolimod has been associated with several safety concerns including bradycardia, AV blocks, increased blood pressure and macular edema.⁷ Based on earlier studies in healthy volunteers, the pharmacodynamic effects of mocravimod included a dose dependent transient reduction in heart rate within the first days after starting treatment and a dose dependent reduction in peripheral lymphocytes (mode of action). With continued daily dosing, the heart rate effect was attenuated and eventually disappeared. In contrast, the reduction in blood lymphocytes is sustained during continuous dosing and is likely explained by persistent internalization of the S1P1 receptor.

In accordance with in vitro findings, two studies (PKRPH001 and PKRPH002) were designed to identify potential drug interactions with mocravimod and mocravimod-phosphate as victims. The objective of these studies was to evaluate the magnitude of the DDIs of mocravimod and mocravimod-phosphate with drugs that affect CYP3A4/P-gP metabolism (itraconazole)^8,9 and inhibit CYP3A4, OATP1B1/B3, P-gp, or BCRP transporters (cyclosporin¹⁰) in healthy subjects. In addition, the reduction in heart rate was assessed after mocravimod treatment.

From day 1 until day 13 of period 2, itraconazole 200 mg was administered once daily (o.d.) in fed conditions. On day 5 of period 2 (P2D5), subjects received oral mocravimod in fed conditions, 60 min after itraconazole dosing. Doses of 200 mg itraconazole (o.d.) were selected as doses of 200 mg to 400 mg are commonly used in therapy. Itraconazole (200 mg o.d.) administration started 4 days prior to evaluating its DDI effect with mocravimod based on regulatory feedback to ensure sufficient plasma levels of itraconazole were reached to allow for evaluating the inhibitory effect of itraconazole.

A follow-up evaluation was performed at the end-of-study (EOS) visit, 25 ± 2 days after P2D5 or in case of early termination. In period 1 and 2, blood samples were collected for determination of mocravimod and mocravimod-phosphate concentrations at pre-dose and 0.25, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, 72, 96, 120, 144 (period 1 only), 168 (period 2 only), 192, 240, 312, and 360 h (period 2 only) post-dose.

Cyclosporin DDI study (PKRPH001). The study design is displayed in Figure S2. The study consisted of a screening period between 28 days and 2 days before the first study treatment administration. On day 1 of period 1, male subjects received a single oral 3 mg dose of mocravimod alone in fasted conditions. Female subjects were not included in this study based upon request from the study center. The rationale for excluding females was based on the label of cyclosporin where teratogenic effects in animals are described, albeit sufficient safety margin compared to the dose in humans.⁸

There was a washout period of at least 4 weeks and up to a maximum of 6 weeks between the two treatment periods. From day 1 until day 7 of period 2, cyclosporin 100 mg was administered orally twice daily, in the morning and evening, 12 h apart, in fed conditions, except for the morning doses on days 1 and 2 when oral cyclosporin was administered in fasted conditions. On day 2 of period 2 (P2D2), subjects received 3 mg oral mocravimod in fasted conditions at the same time as the oral cyclosporin morning dose. A follow-up evaluation was performed at the EOS visit, 42 to 49 days after P2D2 or in case of early termination. In periods 1 and 2, blood samples were collected for determination of mocravimod and mocravimodphosphate concentrations at pre-dose and at 0.25, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, 72, 96, 120, 144, 192, 240, 312, and 360 h (period 2 only) post dose.

Sample Size

In a previous study in healthy subjects, the following intra-participant variabilities (percent coefficient of variation [CV%]) for mocravimod were observed for the relevant exposure measures: AUC0–∞ 9% and C_max 11%. Based on these variabilities, an overall power of 90%, a type 1 error of 5% and allowing for a 5% deviation of the geometric mean ratio (GMR) from the theoretical ratio of 1.0, a projected sample size of 12 participants was calculated to be necessary to be included in the study according to current regulations. Even with a variability of up to 20% in, for example the AUC0–∞ parameter, the power of the study to detect a difference between treatments, if any, was then still >80%. Twenty participants were to be included to allow for the completion of 18 participants and drop-outs were only replaced from the 3rd withdrawn participant to ensure 18 completed participants.

Pharmacodynamics

Heart rate measurements were assessed in the itraconazole DDI study at the following time points: period1: day-1, day 1 pre-dose, 1 h post dose, 6, 12, 24 (day 2), and 48 h (day 3), then on day 5, 7, 9, 11, and 14 in the morning; period 2: day-1, day 5 pre-dose, 1 h post dose, 6, 12, 24 (day 6), and 48 h (day 7) then on day 9, 11, 13, 15, 18, and 20 in the morning.

Heart rate measurements were assessed in the cyclosporin DDI study at the following time points: period 1: day-1, day 1 pre-dose, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, and 12 h post-dose, and then 24 h (day 2), 48 h (day 3) then on day 5 until day 14 in the morning; period 2: day-1, day 2 pre-dose, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, and 12 h post-dose, then 24 h (day 3), and 48 h (day 4) then on day 5 until day 17 in the morning. All vital signs measurements were conducted in supine position after the subject had rested for at least 5 min in the supine position. Nadir of heart rate (PR_nadir(0-24)) and area under the effect curve (AUE_(0-24)PR) were calculated based on trapezoidal rule on nominal times per individual.

Statistical Analysis

The statistical analyses were performed using SAS software version 9.4 (SAS Institute Inc., Cary, NC, USA). PK parameters after administration of mocravimod and mocravimod-phosphate measured in presence and in absence of itraconazole or cyclosporin was analyzed in agreement with the FDA guidance document.¹¹ The effect of concomitant administration of mocravimod with itraconazole or cyclosporin was assessed using the geometric mean ratio and 90% confidence intervals (CIs) of mocravimod and mocravimod-phosphate blood PK parameters C_max and AUC_0-inf. A linear mixed model with a fixed effect for treatment and a random effect for subject was used for natural log-transformed C_max and AUC_0-inf. Geometric leastsquares means were provided for each treatment. In all comparisons, mocravimod administered alone was used as the reference. For the interferential analysis of safety data, the comparison between mocravimod and mocravimod + cyclosporin was conducted. The PR parameters (PR_nadir(0-24) and AUE(0-24)PR), parameters in the cyclosporin DDI study were analyzed by arithmetic means of a mixed effects model, and included treatment as factor, pre-dose value as covariate and subject as a random effect. The mean was estimated for each treatment group. The mean differences between treatment means, was also estimated, along with its 90% CI.

In case a relevant PK interaction was found based on GMR result, further exploratory covariate analyses would be conducted. Therefore, the impact of itraconazole and cyclosporin on relative bioavailability, absorption, and clearance was explored to evaluate the potential role of CYP3A4, P-gp, and BCRP inhibition by means of covariate population PK modeling.¹³ This exploratory evaluation and quantification of the net effect of CYP3A4, P-gp, or BCRP inhibition on PK parameters were conducted in a step-wise approach. Firstly, the relationship between individual random effects after dosing of mocravimod and mocravimodphosphate in absence and presence of cyclosporin and itraconazole were explored in a graphical exploratory analysis. Secondly, if trends and correlations between estimated individual random effects of mocravimod and mocravimod-phosphate in the absence and presence of cyclosporin/itraconazole were detected, exploratory covariate model development was performed. Following the assumption that itraconazole is a P-gp and CYP3A4 inhibitor, whilst cyclosporin is inhibiting P-gp, CYP3A4, and BCRP,¹⁴ covariate effects on clearance and absorption were considered. The presence/absence of itraconazole/cyclosporin was modeled as a categorical covariate. Per default, itraconazole was considered as a covariate, which is not time-varying over the time-course that follows a dosing event. As the data exploration did not indicate that the inhibitory effects of cyclosporin drop significantly in a phase where the elimination of mocravimod and mocravimod-phosphate is still substantial, cyclosporin as time-invariant covariate was used.

Non-linear mixed effects modeling (NONMEM) (version 7.5.1) was used. The “stochastic approximation expectation maximization” (SAEM) method in NONMEM was used as the main parameter estimation algorithm. After each SAEM estimation step, the objective function was determined using importance sampling with the setting EONLY = 1 and MAPITER = 0, as suggested in [ICON 2017].¹⁵

Table 1. Demographics and baseline characteristics of enrolled subjects in the ITZ and CsA DDI studies.

CsA: cyclosporine; DDI: drug–drug interaction; ITZ: itraconazole; N: number of subjects; SD: standard deviation.

Based upon the GMR results, the effect of covariates upon the interaction between cyclosporin and mocravimod required further investigation.

The covariate effect of cyclosporin on clearance and zero-order bioavailability (F_abs0) was evaluated for mocravimod (Table S3). The mocravimod model with cyclosporin effect on F_abs0 resulted in the best Bayesian information criterion value and it was possible to estimate the covariate effect adequately (see model 3, Table S3). The estimated value of the relative bioavailability under the influence of cyclosporin coadministration was increased by an approximate 18% (95% confidence interval: 8% to 29%) for mocravimod.

For mocravimod-phosphate, the effect of cyclosporin on relative bioavailability and the time of the zero-order transfer process were evaluated. The estimated effect of cyclosporin on the bioavailability is ∼59%. However, the model showed instability in the trajectories of the objective function value and the estimated effect is higher than suggested by the bioequivalence testing (results not shown).

During the mocravimod 3 mg period, 25 subjects (100%) had TEAEs that were considered possibly drug-related (arrhythmia supraventricular [1 subject], atrioventricular block first degree [7 subjects, 28%], atrioventricular block second degree [1 subject, 4%], bradycardia [22 subjects, 88%], chest discomfort [1 subject, 4%], lymphocyte count decreased [19 subjects, 76%], and dizziness postural [1 subject, 4%]). During the mocravimod 3 mg + itraconazole 200 mg o.d. period, 18 subjects (94.7%) had TEAEs that were considered as being possibly drugrelated (atrioventricular block first degree [4 subjects, 21%], bradycardia [16 subjects, 84%], lymphocyte count decreased [13 subjects, 68%], dizziness [1 subject, 5%], and headache [1 subject, 5%]). All TEAEs were recovered without sequelae except for one TEAE of itchy nipples which was considered as being not related to study treatment. This event was observed to be recovering/resolving at the EOS visit. Additionally, one TEAE of mild bradycardia was recovered/resolved with sequelae (low pulse rate) at the EOS visit.

No clinically relevant findings were reported from physical examination, vital signs, ECG, and laboratory values except for lymphocyte count and pulse rate where relevant changes were noted in both periods.

In the cyclosporin DDI study, 1 subject withdrew from the study due to an AE of atrioventricular block second degree, considered possibly related to mocravimod. The majority of TEAEs in both periods were of mild or moderate toxicity grade (grade 1 or 2). One AE of “lymphocyte count decreased” was reported as severe (grade 3) toxicity grade in the mocravimod 3 mg + cyclosporin 100 mg twice daily period. During the mocravimod 3 mg period, 21 subjects (100%) had TEAEs that were considered possibly drug-related (atrioventricular block second degree [1 subject, 5%], bradycardia [21 subjects, 100%], asthenia [1 subject, 5%], and lymphocyte count decreased [9 subjects, 43%]). During the mocravimod 3 mg + cyclosporin 100 mg twice daily period, 19 subjects (100%) had TEAEs that were considered possibly drug-related (atrioventricular block first degree [1 subject, 5%], bradycardia [19 subjects, 100%], headache [3 subjects, 16%], soft feces [1 subject, 5%], and lymphocyte count decreased [10 subjects, 53%]). All TEAEs were recovered/resolved without sequelae by the EOS visit.

In the cyclosporin DDI study, at baseline for period 1, median (range) lymphocytes were 1.870 (1.42 to 2.71) × 109/L. At P1D2 median lymphocytes dropped to 1.040 (0.58 to 1.73) × 109/L. At baseline for period 2, median (range) lymphocytes were 1.950 (1.47 to 2.61) × 109/L, with a lower value of 0.890 (0.46 to 1.62) × 109/L at P2D3. Median lymphocyte levels returned to baseline levels by EOS. Figure 2 illustrates the changes in absolute lymphocyte count for both studies.

Pharmacodynamics

The effect on the heart rate reduction after mocravimod administration was also investigated in the cyclosporin DDI. The arithmetic means of supine heart rate for the first 24 h post-dose in the cyclosporin DDI Study are displayed in Figure S3. Some variability can be seen with and without cyclosporin co-administration. A summary of descriptive supine heart rate parameters in the cyclosporin DDI Study is presented in Table S4. The effect of co-administration of cyclosporin on top of mocravimod showed a small difference in PR_nadir (nadir of heart rate) of −0.58 (90%CI −1.66 to 0.50) bpm, indicating lack of additional effect of combination treatment. In contrast to the AUE_0-24, (area under the effect curve from 0 to 24 h) where a 13.96 (h*bpm) (90% CI −18.89 to 46.82) difference was found for mocravimod alone. However, given the large spread of 90% CI, these results should be interpreted with caution. Both parameters do not show increased risk of heart rate reduction in combination with cyclosporin.

Figure 2. Lymphocytes count (×109/L). ITZ DDI study (N = 25), CsA DDI study (N = 21)

AE: adverse event; b.i.d.: twice daily; CsA: cyclosporine; DDI: drug–drug interaction; ITZ: itraconazole;MOC:mocravimod; o.d.: once daily.

N = number of subjects studied.

A subject with multiple AEs is counted only once in the “any system organ class” row.

A subject with multiple AEs within a primary system organ class is counted only once for that system organ class & treatment.

Treatment-emergent is defined as an AE that started after the first dose of each period up to 27 days (ITZ DDI Study) or 49 days (CsA DDI Study) after the last actual administration of each period (and before the start of period 2 for period 1).

The parameters relative bioavailability and apparent clearance, which finally determine the AUC_0-inf, had very limited shrinkage indicating the reliability of the estimated individual parameters, based on which the AUC_0-inf was derived. Hence, the population PK modeling allowed for a reliable characterization of the individual exposures required for determination of the geometric mean ratio for the AUC_0-inf.

In a subsequent step, covariate modeling was explored on the PK parameters. The goal of the covariate modeling was to build a model that is able to quantify the co-medication effect on the parameters, given the quality and quantity of data of both studies. Therefore, the previous mocravimod and mocravimod-phosphate models were simplified. As the visualization of the absorption phase revealed that the fed subjects have larger mocravimod and mocravimod-phosphate concentrations than the fasted ones, the covariate effect of the fasted prandial state on relative bioavailability for mocravimod, and on relative bioavailability and lag time for mocravimod-phosphate were quantified. Moreover, it was found that for mocravimod, cyclosporin increases relative bioavailability. The effects of cyclosporin on relative bioavailability that were quantified in the covariate modeling for mocravimod
are in line with the results from the bioequivalence testing. As mocravimod is the parent compound and mocravimod-phosphate is the metabolite, thus mocravimod converts to mocravimod-phosphate, it implies that the covariate effect of cyclosporin on relative bioavailability observed for mocravimod is present for mocravimod-phosphate as well. Additional clinical PK data would be needed to stabilize the model for mocravimod-phosphate to further investigate the covariate effects on the PK parameters. Given the limited drug–drug interaction that was observed, its relevance is minimal and therefore no additional modeling steps were pursued. This will be conducted once a larger dataset in patients is available and PK data can be linked to relevant safety and efficacy to inform decision making on, for example dose selection.

Overall, when compared to mocravimod 3 mg administered alone, mocravimod 3 mg co-administered with itraconazole 200 mg o.d. or cyclosporin 100 mg twice daily. had a comparable safety profile. The mostcommonly reported AEs, (bradycardia, decreased lymphocyte count) were consistent with the known adverse reaction of mocravimod 3 mg. A decreased lymphocyte count was expected based on the known characteristic of mocravimod leading to egress of lymphocytes into secondary lymphoid organs and reducing blood lymphocyte counts. This is known to be caused by functional antagonism at the S1P1 receptor on lymphocytes such as T and B cells because of persistent ligand-induced internalization of the S1P1 receptor. The internalization of S1P1 receptors renders these cells unresponsive to S1P, depriving them of an obligatory signal for egress. No SAEs and no fatal TEAEs were reported during these studies.

Transient bradycardia and atrioventricular (AV) blocks are associated with the class of S1P modulators. Most of the labels of approved S1P1 modulators such as fingolimod, ponesimod, estrasimod and siponimod, show clear guidance on first dose effects on heart rate, monitoring guidelines and treatment initiation recommendations.

Heart rate effect for combination with cyclosporin does not show differences (PRnadir of −0.58 (90%CI − 1.66 to 0.50) bpm). Reduction in heart rate is similar to observed changes in heart rate for other S1P modulators such as siponimod or fingolimod.¹⁶ The maximum heart rate effect of the active treatment relative to placebo at doses producing approximately 70% reduction in absolute lymphocyte counts varied across molecules and ranged from approximately −21 to −6.5 bpm for siponimod and −11 to −6 bpm for fingolimod.¹⁶

These results are comparable with the first dose reduction in heart rate observed for several S1P1R modulators.¹⁶

Conflicts of Interest

All authors are employees of Priothera SAS.

Funding

These studies were funded by Priothera SAS.

Data Availability Statement

Priothera SAS will provide access to individual deidentified participant data upon request. To gain access, data requestors must enter into a data access agreement with Priothera SAS.

Ethics Statement

The study protocols, amendments, and informed consent forms were reviewed by an independent ethics committee (Ethic Committee of Ile-de-France VII). These studies were conducted in accordance with the ethical principles in the Declaration of Helsinki and are consistent with International Conference on Harmonisation, Good Clinical Practice and applicable regulatory requirements. All subjects provided written informed consent for study participation before any procedures were performed.

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